Cooperative transition between open and closed conformations in potassium channels

Potassium (K+) ion channels switch between open and closed conformations. The nature of this important transition was revealed by comparing the X-ray crystal structures of the MthK channel from Methanobacterium thermoautotrophicum, obtained in its open conformation, and the KcsA channel from Streptomyces lividans, obtained in its closed conformation. We analyzed the dynamic characteristics and energetics of these homotetrameric structures in order to study the role of the intersubunit cooperativity in this transition. For this, elastic models and in silico alanine-scanning mutagenesis were used, respectively. Reassuringly, the calculations manifested motion from the open (closed) towards the closed (open) conformation. The calculations also revealed a network of dynamically and energetically coupled residues. Interestingly, the network suggests coupling between the selectivity filter and the gate, which are located at the two ends of the channel pore. Coupling between these two regions was not observed in calculations that were conducted with the monomer, which emphasizes the importance of the intersubunit interactions within the tetrameric structure for the cooperative gating behavior of the channel.     

Metal-induced sequential transitions among DNA conformations

The action of [Co(NH₃)₆]Cl₃ on poly(dGdC) · poly(dGdC) can lead to a series of consecutive reactions, in which B-DNA is first converted to Z-DNA [M. Behe and G. Felsenfeld (1981) Proc. Natl. Acad. Sci USA 78 , 1619–1623], which in turn is transformed into an unidentified conformer that we tentatively call “U”, and finally the highly associated anisotropic ψ-DNA is produced. Conditions are given under which the sequence B ? Z ? “U” ψ(+) can be stopped at any point in the direction from left to right. The reverse processes, from right to left, occur when ψ(+) or “U”-DNA are treated with various amounts of salt concentrations and lowering temperature. Thus it is demonstrated that four conformers of poly(dGdC) · poly(dGdC) are readily interconvertible, and that Z-DNA and “U” conformers are intermediates in the reversible transformations of B- and ψ-DNA.     

Equilibrium transitions between side‐chain conformations in leucine and isoleucine

Despite recent improvements in computational methods for protein design, we still lack a quantitative, predictive understanding of the intrinsic probabilities for amino acids to adopt particular side‐chain conformations. Surprisingly, this question has remained unsettled for many years, in part because of inconsistent results from different experimental approaches. To explicitly determine the relative populations of different side‐chain dihedral angles, we performed all‐atom hard‐sphere Langevin Dynamics simulations of leucine (Leu) and isoleucine (Ile) dipeptide mimetics with stereo‐chemical constraints and repulsive‐only steric interactions between non‐bonded atoms. We determine the relative populations of the different χ₁ and χ₂ dihedral angle combinations as a function of the backbone dihedral angles ? and ψ. We also propose, and test, a mechanism for inter‐conversion between the different side‐chain conformations. Specifically, we discover that some of the transitions between side‐chain dihedral angle combinations are very frequent, whereas others are orders of magnitude less frequent, because they require rare coordinated motions to avoid steric clashes. For example, to transition between different values of χ₂, the Leu side‐chain bond angles κ₁ and κ₂ must increase, whereas to transition in χ₁, the Ile bond angles λ₁ and λ₂ must increase. These results emphasize the importance of computational approaches in stimulating further experimental studies of the conformations of side‐chains in proteins. Moreover, our studies emphasize the power of simple steric models to inform our understanding of protein structure, dynamics, and design. Proteins 2015; 83:1488–1499. © 2015 Wiley Periodicals, Inc.     

CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome

Conformational change within the spliceosome is required between the first and second catalytic steps of pre-mRNA splicing. A prior genetic screen for suppressors of an intron mutant that stalls between the two steps yielded both prp8 and non-prp8 alleles that suppressed second-step splicing defects. We have now identified the strongest non-prp8 suppressors as alleles of the NTC (Prp19 complex) component, CEF1. These cef1 alleles generally suppress second-step defects caused by a variety of intron mutations, mutations in U6 snRNA, or deletion of the second-step protein factor Prp17, and they can activate alternative 3' splice sites. Genetic and functional interactions between cef1 and prp8 alleles suggest that they modulate the same event(s) in the first-to-second-step transition, most likely by stabilization of the second-step spliceosome; in contrast, alleles of U6 snRNA that also alter this transition modulate a distinct event, most likely by stabilization of the first-step spliceosome. These results implicate a myb-like domain of Cef1/CDC5 in interactions that modulate conformational states of the spliceosome and suggest that alteration of these events affects splice site use, resulting in alternative splicing-like patterns in yeast.     

Surface active properties of amphiphilic sequential isopeptides: Comparison between alpha-helical and beta-sheet conformations

Poly(Leu-Lys-Lys-Leu) and poly(Leu-Lys) are sequential amphiphilic peptide isomers that adopt respectively an alpha-helical conformation and a beta-sheet structure in saline solutions and at the air/water interface. The surface active properties of LKKL and LK sequential isopeptides containing 16, 20, and n residues have been compared in order to evaluate the contributions of the alpha-helical and beta-sheet conformations. Both have a natural tendency to spread at the surface of a saline solution and the values of the equilibrium spreading pressure pi(e) lie in the same range. When dissolved in a saline solution, alpha-helical peptides diffuse faster and adsorb faster at the interface than the beta-sheet isomers. From the compression isotherms of LKKL and LK peptide monolayers it is possible to extract parameters that characterize the behavior of alpha-helical and beta-sheet conformations: beta-sheet peptide monolayers are more stable and less compressible than the monolayers formed with the alpha-helical isomers. The LK peptides differ also by their high degree of self-association at the air/water interface. Copyright 1999 John Wiley & Sons, Inc.     

Cooperative binding between distant transcription factors is a hallmark of active enhancers

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Dynamic equilibrium between multiple active and inactive conformations explains regulation and oncogenic mutations in ErbB receptors

The ErbB growth factor receptor family members are key players in vital physiological and pathological processes. Like other receptor tyrosine kinases, the ErbBs are bi-topic membrane proteins, whose extracellular and intracellular domains are connected by single transmembrane span. In recent years the crystal structures of the extracellular and intracellular domains of some ErbBs have been determined. We integrated the available structural information with phylogenetic, biochemical, biophysical, genetic, and computational data into a suggested model for the regulation and activation of these receptors. According to the model, regulation is maintained by a dynamic equilibrium between monomeric and dimeric states in various conformations. Along this dynamic equilibrium, variations in the points of interactions within the dimers alter the activation state and ligand-binding affinities. The active state was recently shown to be associated with an asymmetric dimer of the kinase domains. That finding enabled us to elucidate, in molecular terms, the directionality observed in the activation process of ErbB heterodimers; it can explain, for example, the preferential activation of ErbB2 by ErbB1 over activation of ErbB1 by ErbB2. Sequence alterations that reverse this directionality lead to aberrant signaling and cancer. Our model also offers molecular interpretations of the effects of various oncogenic alterations that interfere with the regulatory mechanism.     

Salt induced transitions between multiple conformations of poly (rG-m5dC).poly (rG-m5dC).

Salt induced transitions between four conformations of the methylated ribo-deoxyribo co-polymer poly (rG-m5dC).poly (rG-m5dC) have been studied using phosphorous-NMR, Raman spectroscopy, and circular dichroism. A high salt A-Z transition is observed for the polymer. However, the methylated polymer does not enter the high salt Z form more readily than the analogous unmethylated polymer, unlike the effect of methylation on the fully deoxy polymer poly (dG-dC).poly (dG-dC). The methylated polymer fails to undergo a low salt A-Z transition in 5 mM Tris buffer, unlike the unmethylated poly (rG-dC).poly (rG-dC). However, if the counterion is changed to triethanolamine buffer, an A-Z transition does take place. In 5 mM Tris buffer the phosphorous-NMR spectrum of poly (rG-m5dC).poly (rG-m5dC) shows one resonance in the absence of NaCl that splits into two closely spaced resonances as the NaCl level is increased to 30 mM. The Raman spectrum of poly (rG-m5dC).poly (rG-m5dC) shows that it is in the A conformation at intermediate salt concentrations. From this we conclude that poly (rG-m5dC).poly (rG-m5dC) is in a regular A conformation in Tris buffer at low Na+ levels, shifting to an alternating A conformation with a dinucleotide repeat at intermediate salt concentrations.     

Differential spatial approximation between secretin and its receptor residues in active and inactive conformations demonstrated by photoaffinity labeling

Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([psi(4, 5)]secretin) in which the peptide bond between residues 4 and 5 was replaced by a psi(CH(2)-NH) peptide bond isostere. We have developed a series of [psi(4, 5)]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.     

Phase transitions in 1,2-and 1,3-diacyl-sn-glycerol-3-phosphocholine monolayers at the oil/water interface

Moving the phosphatidylcholine group from the 3-to the 2-position in monolayers of $\text{distearoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

Biologically active conformations of thymopentin. Studies with conformationally restricted analogs

Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.     

An RNA secondary structure switch between the inactive and active conformations of the Escherichia coli 30 S ribosomal subunit

Psoralen cross-linking was used to produce intramolecular cross-links in the Escherichia coli 16 S ribosomal RNA in the inactive and active forms of the 30 S subunit. A number of psoralen cross-links were made in the inactive form that were not made in the active form. The most frequent of these cross-links was sequenced by a series of techniques and identified as C-924 to U-1532. In this region, a three-base complementary, (921-923).(1532-1534), forms a site where psoralen can stack and produce a cross-link between C-924 and U-1532. When psoralen monoadducts were placed on inactive subunits and the conformation was switched to the active form before cross-linking, a new cross-link involving U-1393 was detected. U-1393 is part of the complementarity, (923-925).(1391-1393), that has previously been proposed as being an element of the functional secondary structure on the basis of sequence comparison. The complementarity between (921-923).(1532-1534) occurs in most nonmitochondrial small subunit RNAs; however, there are several counter examples in which it does not occur. This suggests that this alternate secondary structure interaction is not necessary for the function of the 30 S subunit.     

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.     

Crystal structures of a Rab protein in its inactive and active conformations

We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural features common to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.     

The switch between two conformations of adenylate kinase

Crystalline adenylate kinase from porcine muscle cytosol can assume two interconvertible structures. Here, we report the refined structure of crystal form B at 3.3 A resolution and compare it with crystal form A. Crystal forms A and B can be interconverted by protonation and deprotonation of His36, which is located deep in the active center cleft. The changes concern the molecular packing as well as the polypeptide chain conformation. On conversion from crystal form A to B, the N-terminal alpha-helix unwinds, the active center cleft opens to some extent and the nucleotide-binding glycine-rich loop 15-22 at the active center is detached from the bulk protein. This loop has counterparts in various important mononucleotide-binding proteins and is known to bind a phosphoryl group in adenylate kinase and in the oncogenic ras proteins. It is most likely involved in the phosphoryl transfer and the concomitant conformational changes. it is suggested that the two observed conformations are relevant for enzyme action in solution: they represent two of a series of three known snapshots depicting the enzyme during the substrate binding process.     

Multiple active site conformations revealed by distant site mutation in ornithine decarboxylase

Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle.